Protease for wound conditioning and skin care

ABSTRACT

We have identified by molecular cloning a protease which originates from the larvae of Lucilia sericata and which was termed debrilase due to its activities useful for debridement of wounds. Described is a nucleic acid molecule encoding a serine protease having the ability to cleave fibrin and casein which is (a) a nucleic acid molecule encoding the serine protease comprising or consisting of the amino acid sequence of SEQ ID NO: 4 as well as to nucleic acid molecules encoding precursors or fragments of said serine protease; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3; (c) a nucleic acid molecule encoding a serine protease the amino acid sequence of which is at least 80% identical to the amino acid sequence of (a), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 80% identical to the nucleotide sequence of (b), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (b) or (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.

RELATED APPLICATIONS

This application is a reissue application of U.S. Pat. No. 8,623,810,issued on Jan. 7, 2014, from U.S. patent application Ser. No.13/245,157, filed on Nov. 17, 2011, which is the National Phase ofinternational Application No. PCT/EP2010/001328, filed Mar. 3, 2010,which designated the U.S. and that International Application waspublished under PCT Article 21(2) in English, and claims priority toEuropean Application No. 09003063.6, filed Mar. 3, 2009, all of whichapplications are incorporated herein by reference in their entirety.

The invention relates to pharmaceutical and cosmetical compositions aswell as medical articles comprising a protease, termed debrilase, fromLucilia sericata for use in wound conditioning and skin care. Debrilasewas identified using the larval transcriptome, i.e. mRNA preparationsand conversion into cDNA, and prepared using recombinant techniques andsubsequent expression in suitable host cells. The protease of theinvention has fibrinolytic, ca seinolytic and PAR-2 (protease-activatedreceptor 2) activating activity. In one embodiment, compositionscomprising debrilase are provided for the cleaning of slow ornon-healing wounds from necrotic cells, tissue and fibrin cuffs, knownas debridement. In another embodiment cosmetic uses of debrilase in thefield of skin care and smoothening are proposed.

The invention further relates to a nucleic acid molecule encoding aserine protease having the ability to cleave fibrin and casein, which isa nucleic acid molecule encoding the serine protease comprising orconsisting of the amino acid sequence of SEQ ID NO: 4 as well as tonucleic acid molecules encoding precursors or fragments of said serineprotease. Furthermore, it relates to a nucleic acid molecule comprisingor consisting of the nucleotide sequence of SEQ ID NO: 3 and a nucleicacid molecule encoding a serine protease the amino acid sequence ofwhich is at least 77%, more preferred at least 80%, even more preferredat least 85% and most preferred at least 90% identical to the nucleotidesequence of SEQ ID NO: 3. Any of the above nucleic acids, wherein T isreplaced by U are also within the scope of the invention.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Wound healing has three distinct phases: (1) inflammation; (2) cellmigration and proliferation; and (3) remodelling. In the inflammatoryphase proteases are released by cells of the immune system. Variouslymphokines are secreted from neutrophils and macrophages that modulatethe next phase of the wound healing. The second phase is designatedproliferation phase and includes fibroblast migration, proliferation andthe synthesis of new extracellular matrix molecules. These events appearto occur in a definite order where extracellular matrix moleculesincluding fibronectin, collagen and proteoglycans are secreted into thegranulation bed. The inflammatory phase peaks at 3 days. The secondphase of wound healing normally peaks at approximately one to two weeksafter injury and is followed by a much longer third phase of tissueremodelling that begins within weeks and may last several months. Duringthe remodelling phase, the connective tissue matrix matures as thedisorganized collagen fibers are replaced by thicker, more alignedcollagen molecules. This tissue remodelling eventually contributes tothe tensile strength of the wound and is sometimes accompanied by scarformation.

A skin lesion virtually always involves an injury of the blood vessels.Wound healing therefore also comprises the process of coagulation, beinga complex process by which blood forms clots. It is an important part ofhemostasis whereby a damaged blood vessel wall is covered by a plateletand fibrin containing clot to stop bleeding and begin repair of thedamaged vessel. Disorders of coagulation can lead to an increased riskof hemorrhage and/or thrombosis. Coagulation is initiated almostinstantly after an injury to the blood vessel has damaged theendothelium. Platelets immediately form a hemostatic plug at the site ofinjury (primary hemostasis). Secondary hemostasis occurs simultaneouslywhere coagulation factors in the blood plasma respond in a complexcascade to form fibrin strands which strengthen the platelet plug. Thecoagulation cascade of secondary hemostasis has two pathways, thecontact activation pathway (formerly known as the intrinsic pathway) andthe tissue factor pathway (formerly known as the extrinsic pathway) bothleading to fibrin formation. It is now known that the primary pathwayfor the initiation of blood coagulation is the tissue factor pathway.The pathways are a series of reactions, in which a zymogen (inactiveenzyme precursor) of a serine protease and its glycoprotein co-factorare activated to become active components that then catalyze the nextreaction in the cascade, ultimately resulting in cross-linked fibrin.The coagulation factors are generally serine proteases, except for FVIIIand FV which are glycoproteins and Factor XIII which is atransglutaminase. The coagulation cascade is classically divided intothree pathways. The tissue factor and the contact activation pathwayboth activate the “final common pathway” of factor X, thrombin andfibrin.

Fibrin is a protein involved in the clotting of blood. It is a fibrillarprotein that is polymerised to form a hemostatic plug or clot—inconjunction with platelets—over a wound site. Fibrin is made from itszymogen fibrinogen, a soluble plasma glycoprotein that is synthesised bythe liver. In the coagulation cascade the zymogen prothrombin isactivated to the serine protease thrombin, which is responsible forconverting fibrinogen into fibrin. Fibrin is then cross linked by factorXIII to form a clot.

Plasmin proteolytically cleaves fibrin into fibrin degradation productswhich inhibits excessive fibrin formation. Plasmin is generated byproteolytic cleavage of plasminogen, a plasma protein synthesized in theliver. This cleavage is catalyzed by tissue plasminogen activator (t-PA)which is synthesized and secreted by the endothelium. Plasminogen isentrapped during clot formation and is slowly activated when the woundhas stopped bleeding and the clot is broken down.

The disintegration of the clot during wound healing is an important stepto allow for matrix formation and remodelling as described above. It isthought that one potential cause for slow healing or chronic wounds is alack of fibrinolysis. So called “fibrin cuffs”, consisting of fibrin,laminin, fibronectin, tenascin, collagen and leucocytes hamper theexchange of nutrients, growth factors and gas, leading to anoxia, ulcusformation and preventing angiogenesis. The proteolytic dissolution ofthese “fibrin cuffs” supports neovascularisation, invasion ofleucocytes, migration of fibroblasts, formation of a new epithelium andinduces cell proliferation and migration. Furthermore, the healing ofwounds is delayed by the presence of pus, tissue debris, bacteria, andexudates which can be removed by debridement agents.

Debridement is defined as the removal of non-vital tissue from wounds.In chronic wounds, debridement means the elimination of necrosis as wellas the clearing away of wound dressings, foreign bodies, and othernon-vital substances. Sufficient debridement represents one basicprerequisite for a non-delayed wound-healing process. In addition totreating the causal factors for delayed wound healing, debridementshould be the first step in an adequate phase-adapted wound-bedpreparation for chronic wounds. Different methods for debridement inchronic wounds have been described such as surgery, maggot therapy,laser, ultrasound, hydrotherapy, wet-to-dry method, autolysis,proteolytic enzymes, osmotic or chemical debridement.

Debridement agents rapidly digest necrotic tissue without injury toliving cells, thereby speeding up the healing processes. The search forsuch debridement agents has included the employment of a wide variety ofplant and animal materials, such as maggots or blowfly larvae, but alsoof enzymes like papain, bromelain and ananain from plant origin (e.g.U.S. Pat. No. 6,548,556, EP 0194647 B1, U.S. Pat. No. 5,106,621),proteases from microbial origin (e.g. protease from Vibrio spec., U.S.Pat. No. 5,145,881; thermolysin from Bacillus spec., WO 03/088993 A1, US2003/0198632 A1) and from animals (e.g. trypsin and chymotrypsin fromfish, U.S. Pat. No. 6,846,485). Some debriding enzymes function asprotease-inhibitors either within the coagulation cascade (thrombininhibitory nexin-1 (PN-1), U.S. Pat. No. 5,112,608) or in theacute-phase-reaction (alpha1-antitrypsin. U.S. Pat. No. 6,262,020). Theprimary purpose of debriding enzymes is to clean a wound of all of thevarious necrotic tissue elements and to thin out tenacious exudativesecretions. Appropriately applied proteolytic enzymes cleanse infectedsurfaces of their inflammatory exudate without harming the livingtissues. They facilitate the drainage of areas of located purvulent,sanguineous and fibrinous accumulations, promote the liberation ofhidden bacteria rendering them accessible to the immune defence system.

The enzymatic action of debriding enzymes can be utilised fornon-healing wounds (e.g. Lobmann et al., Proteases and the diabetic footsyndrome: Mechanisms and therapeutic Implications. Diabetes Care 28(2005), 461-471), but they can also be of benefit for the treatment ofinflammatory skin diseases such as psoriasis and eczema and the like,and less severe skin conditions, such as wrinkles, acne and dry skin, asdisclosed for example in U.S. Pat. Nos. 4,524,136; 5,439,935; 5,441,740;5,554,366; 5,853,705 and 6,780,444. Commercial products comprising suchenzymes are also available, e.g. Accuzyme® (papain) and Granulex®(trypsin), the application of which is limited for debridement ofwounds.

There has been a continuing effort to find better wound debridementenzymes. Some of the criteria for a highly preferred wound debridementenzyme are at least one and preferably more such as all of thefollowing: it should be capable of rapid digestion of fibrin, denaturedcollagen, elastin and exudate; it should spare normal appearing humanskin tissues; it should be non-toxic and non-irritating to wounds; itshould be easily prepared, stable and readily applicable. As an example,stable concentrated enzymatic compositions suitable for storage underambient conditions, while maintaining enzymatic activity and kitscomprising enzymatic compositions are described in WO 2007/074454 A2.

Several approaches for dealing with chronic wounds comprise the use ofdressings keeping the wound moist to support the viability of cells ofthe immune system infiltrating the wound.

For wound management, the use of proteases, i.e. exo- andendopeptidases, as well as the use of protease inhibitors was proposed.

A silicone-based device for controlled release of enzymes for theproteolytic debridement of wounds was described by Bott et al. (Asilicone-based controlled-release device for accelerated proteolyticdebridement of wounds, Wound Repair Regen. (2007) 15: 227-35) using aprotease of the subtilisin family. Similarly, U.S. Pat. No. 7,368,128describes a dressing for sustained release of debriding enzymesconsisting of an absorbent material layer and a degradable polymericmaterial.

For centuries, maggots were known to have beneficial effects on wounds.The method of Maggot Debridement Therapy (MDT) was adopted and routinelyused in the United States throughout the 1930s and early 1940s, but MDTwas replaced with the introduction of penicillin and modern surgicalprocedures (Child F S, Roberts E F. The treatment of chronicosteomyelitis with live maggots. New York State J Med 1931; 31: 937-43;Teich S, Myers R A M. Maggot therapy for severe skin infections. SouthMed J 1986; 79: 1153-5; Church J C T. Larvae therapy in modern woundcare: A review. Primary Intention 1999; May: 63-8; Sherman R A, Hall M JR, Thomas S. Medicinal maggots: An ancient remedy for some contemporaryafflictions. Ann Rev Entomol 45 (2000): 55-81, Courtenay, M. et al.,Larva therapy in wound management, J. R. Soc. Med. 93 (2000): 72-74).

Application of maggots to a wound is frequently perceived as unpleasantand even painful by the patient. Nevertheless, the larvae of differentinsect species are utilised for the cleaning and healing ofconventionally untreatable wounds. Maggots of certain fly species feedon necrotic tissue and through this debriding activity assist thehealing of chronic soft-tissue wounds, such as pressure and venousstasis ulcers, diabetic foot infections, and postoperative wounds, whichare resistant to surgical or antibiotic intervention (Sherman R A, 1998,Maggot debridement in modern medicine. Infect Med 15: 651-6, Sherman RA, Hall M J R, Thomas S., 2000, Medicinal maggots: An ancient remedy forsome contemporary afflictions. Ann Rev Entomol 45: 55-81).

Medicinal maggots exert three main actions: 1) they debride (clean)wounds by dissolving the dead (necrotic), infected tissue; 2) theydisinfect the wound, by killing bacteria; and 3) they stimulate woundhealing.

Horobin et al. (2006) describe MDT with Lucille sericata larvae or greenbottle fly maggots which are applied to chronic wounds to aid healing bytriggering fibroblast migration and matrix remodelling (Horobin et al.,Promotion of human dermal fibroblast migration, matrix remodelling andmodification of fibroblast morphology within a novel 3D model by Luciliasericata larval secretions, J Invest Dermatol. 126: 1410-1418).Previously, they have characterized certain enzymatic activities presentwithin maggot excretions/secretions (ES) (Horobin et al. 2003, Maggotsand wound healing: an investigation of the effects of secretions fromLucille sericata larvae upon interactions between human dermalfibroblasts and extracellular matrix components. Br. J. Dermatol. 148:923-933).

The bactericidal activity of secretions from Lucille sericata maggotstowards typical wound colonizers such as Micrococcus luteus andStaphylococcus aureus was shown using third instar larvae (Daeschlein,G. et al., 2007, In vitro antibacterial activity of Lucilia sericatamaggot secretions, Skin Pharmacol. Physiol. 20: 112-115).

Interactions between fibroblasts and the surrounding extracellularmatrix play a crucial role in tissue formation, influencing fibroblastproliferation, migration, and tissue remodelling. The postulatedmechanisms by which maggots enhance tissue formation within wounds maybe via the promotion of fibroblast motility, acceleration ofextracellular matrix remodelling and coordination of cellular responses,providing for a wider distribution of viable fibroblasts. It was shownthat L. sericata ES-products promoted fibroblast migration upon afibronectin-coated surface and that this was related to the degradationof fibronectin by serine proteases within maggot excretion/secretions(Chambers et al. 2000, Degradation of extracellular matrix components bydefined proteinases from the greenbottle larva Lucilia sericata used forthe clinical debridement of non-healing wounds, Brit J Dermatol. 148:14-23).

A large and diverse family of serine protease genes was identified infirst instar larval cDNA of the sheep blowfly, Lucilia cuprina (Elvin etal. 1994, An estimate of the number of serine protease genes expressedin sheep blowfly larvae (Lucille cuprina)” Insect Molecular Biol 3:105-115). Two chymotrypsin-like proteases were purified from theES-products of first instar larvae of Lucille cuprina (Casu et al. 1994,Excretory/secretory chymotrypsin from Lucilia cuprina: purification,enzymatic specificity and amino acid sequence deduced from mRNA, InsectMolecular Biol 3: 201-211). Various protease inhibitors active againstboth trypsin- and chymotrypsin-like serine proteases were used tocharacterize gut proteases from Lucilia cuprina by in vitro feedingassays (Casu et al. 1994, Isolation of a trypsin-like serine proteasegene family from the sheep blowfly Lucille cuprina, Insect MolecularBiol 3: 159-170). Significant larval growth retardation, was observed onfeeding first instar larvae with trypsin inhibitors, particularlysoybean trypsin inhibitor. Feeding of chymostatin, a specificchymotrypsin inhibitor, resulted in no significant growth retardation.This information suggests that trypsin-like serine proteases areprobably the major gut digestive enzymes.

Chambers et al. (2003, Degradation of extracellular matrix components bydefined proteinases from greenbottle larva Lucilia sericata used for theclinical debridement of non-healing wounds, British J. Dermatol 148:14-23) describe proteases with activities against fibrin andextracellular matrix components in first to third instar larvae of thegreenbottle fly Lucille sericata. They found the highest specificactivity in excretions of the early larval stages (first and secondinstar). Proteases with chymotrypsin and trypsin-like activitiescontained in larval excretory/secretory (ES) products are thought tocontribute to wound debridement by removal of necrotic tissue. Threeclasses of proteolytic enzyme were detected in the secretions usingfluorescein isothiocyanate-labelled casein as a model substrate: serineproteinases (pH optima 8-9) of two different subclasses (trypsin-likeand chymotrypsin-like), aspartyl proteinase (pH optimum 5) and ametalloproteinase (pH optimum 9) with exopeptidase characteristics.Using skin-relevant ECM components as substrates L. sericata ES productssolubilized fibrin clots and degraded fibronectin, laminin andacid-solubilized collagen types I and III.

Despite these results, until today scientists and medical doctors seekfor the active principle behind the huge amount of proteolyticallyactive proteins in the larvae. It is known that larval secretions arecapable of degrading the extracellular matrix (ECM)/wound components,fibronectin, laminin and collagens I, III, IV and V. Thesemacromolecules are found in the slough of chronic wounds and also makeup the “fibrin cuffs” that are predominant in chronic ulcers. Thedegradation of laminin and fibronectin by larval secretions is inhibitedby PMSF, but not significantly by APMSF or by the metalloproteinaseinhibitor 1,10-phenanthroline. The activity of the larval secretionsexhibit a pH optimum of 8.0-8.5 (Chambers et al. 2000, Degradation ofextracellular matrix components by defined proteinases from thegreenbottle larva Lucilia sericata used for the clinical debridement ofnon-healing wounds, Brit J. Dermatol. 148: 14-23), which is notconsistent with the wide pH-range from acidic to basic in chronicwounds. Therefore, there is a need for debriding enzymes with activityin a much more extensive pH-range as that described for the larvalsecretions.

In U.S. Pat. No. 7,144,721 B1 it is speculated that the main proteolyticactivity in larval secretions is a serine proteinase activity and thatthere are two types of serine proteinase activity present; one derivedfrom a chymotryptic enzyme and one derived from a tryptic enzyme. US2008/0108551 A1 provides evidence that degradation of the extracellularmatrix is an important step in protease mediated wound healing byLucilia sericata. In said application a serine protease and ametalloprotease isolated from Lucilia sericata excretory/secretoryproducts are postulated, which degrade the extracellular matrixcomponent fibronectin or increase cell migration, respectively.Apparently, the resulting biologically active degradation products offibronectin promote wound healing. In WO 2007/138361 A3 a chymotrypsinfrom Lucilia sericata larvae for use in the treatment of wounds isdisclosed. Further proteins which are apparently involved in woundhealing like a nuclease (WO 2007/122424 A2) and a ligand for thetoll-like receptor (US 2005/0053597 A1) were identified in the secretionproducts of larvae from Lucilia sericata.

Despite the progress made in recent years in the development ofdebridement compositions, it would still be desirable to provide clearlydefined debridement compounds or compositions comprising definedcomponents with at least similar if not improved properties as comparedto the above discussed prior art materials. This aim could e.g. beachieved by identifying the active principle responsible for improvedwound healing provided by maggots. Such compounds would also be usefulfor cosmetic applications.

Accordingly, in a first embodiment the invention relates to a nucleicacid molecule encoding

(i) a serine protease having the ability to cleave fibrin and casein,which is (a) a nucleic acid molecule encoding the serine proteasecomprising or consisting of the amino acid sequence of SEQ ID NO: 4; (b)a nucleic acid molecule comprising or consisting of the nucleotidesequence of SEQ ID NO: 3; (c) a nucleic acid molecule encoding a serineprotease the amino acid sequence of which is at least 80% identical tothe amino acid sequence of (a); preferably at least 85% identical, morepreferably at least 90% identical, and most preferred at least 95%identical; (d) a nucleic acid molecule comprising or consisting of anucleotide sequence which is at least 80% identical to the nucleotidesequence of (b), preferably at least 85% identical, more preferably atleast 90% identical, and most preferred at least 95% identical; (e) anucleic acid molecule which is degenerate with respect to the nucleicacid molecule of (d); or (f) a nucleic acid molecule corresponding tothe nucleic acid molecule of any one of (a) to (d) wherein T is replacedby U;(ii) a fragment of the serine protease of (i) with the same activity ofthe serine protease of (i);(iii) a propeptide of the serine protease of (i) which is cleaved to itsactive form preferably immediately before or during treatment of awound, wherein the propeptide is encoded by a nucleic acid moleculeselected from (a) a nucleic acid molecule encoding the serine proteasepropeptide comprising or consisting of the amino acid sequence of SEQ IDNO: 6; (b) a nucleic acid molecule comprising or consisting of thenucleotide sequence of SEQ ID NO: 5; (c) a nucleic acid moleculeencoding a serine protease propeptide the amino acid sequence of whichis at least 80% identical to the amino acid sequence of (a); preferablyat least 85% identical, more preferably at least 90% identical, and mostpreferred at least 95% identical; (d) a nucleic acid molecule comprisingor consisting of a nucleotide sequence which is at least 80% identicalto the nucleotide sequence of (b), preferably at least 85% identical,more preferably at least 90% identical, and most preferred at least 95%identical; (e) a nucleic acid molecule which is degenerate with respectto the nucleic acid molecule of (d); or (f) a nucleic acid moleculecorresponding to the nucleic acid molecule of any one of (a) to (d)wherein T is replaced by U; or(iv) a pre-propeptide of the serine protease of (i), wherein thepre-propeptide is encoded by a nucleic acid molecule selected from (a) anucleic acid molecule encoding the serine protease pre-propeptidecomprising or consisting of the amino acid sequence of SEQ ID NO: 2; (b)a nucleic acid molecule comprising or consisting of the nucleotidesequence of SEQ ID NO: 1; (c) a nucleic acid molecule encoding a serineprotease pre-propeptide the amino acid sequence of which is at least 80%identical to the amino acid sequence of (a); preferably at least 85%identical, more preferably at least 90% identical, and most preferred atleast 95% identical; (d) a nucleic acid molecule comprising orconsisting of a nucleotide sequence which is at least 80% identical tothe nucleotide sequence of (b), preferably at least 85% identical, morepreferably at least 90% identical, and most preferred at least 95%identical; (e) a nucleic acid molecule which is degenerate with respectto the nucleic acid molecule of (d); or (f) a nucleic acid moleculecorresponding to the nucleic acid molecule of any one of (a) to (d)wherein T is replaced by U.

The term “nucleic acid molecule” in accordance with the presentinvention includes DNA, such as cDNA or genomic DNA, and RNA. Furtherincluded are nucleic acid mimicking molecules known in the art such assynthetic or semi-synthetic derivatives of DNA or RNA and mixedpolymers. Such nucleic acid mimicking molecules or nucleic acidderivatives according to the invention include phosphorothioate nucleicacid, phosphoramidate nucleic acid, 2′-O-methoxyethyl ribonucleic acid,morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleicacid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, ChemBiol 2001, 8: 1). LNA is an RNA derivative in which the ribose ring isconstrained by a methylene linkage between the 2′-oxygen and the4′-carbon.

The term “serin protease” characterizes proteins having proteolyticactivity as belonging to a subgroup the members of which comprise aserine in their active centre which together with histidine andaspartate forms the catalytic triad common to most serine proteases(Rawlings, N. D., Barrett, A. J. (1994). Families of serine peptidases.Meth. Enzymol. 244:19-61). Serine-proteases are classified as hydrolasesand belong to the EC numerical classification scheme subgroup 3.4.21.The proteins encoded by the nucleic acid molecule of the invention mayeither themselves exhibit the proteolytic activity of a serine protease(e.g. SEQ ID NO: 4) or after further maturation or activation (e.g. SEQID NO: 2 or 6), e.g. by proteolytical processing; see also thediscussion of the propeptide of the invention below. Proteolyticalprocessing may include the cut-off of a signal peptide from apre-propeptide (e.g. the pre-propeptide having SEQ ID NO: 2) and thefurther proteolytical processing of the propeptide (e.g. the propeptidehaving SEQ ID NO: 6) by proteolytic cleavage.

The term “signal peptide” as used herein defines a short amino acidsequence (preferably, 3-60 amino acids long) that directs the transportof a protein to a particular cell compartment and preferably to theendoplasmic reticulum.

The term “propeptide” as used herein describes a linear molecular chainof amino acids, which is a precursor of a protein and is cleaved duringmaturation or activation of the protein (e.g. the amino acid sequence ofSEQ ID NO: 6 or a amino acid sequence encoded by the nucleotide sequenceof SEQ ID NO: 5). The term “pre-propeptide” as used herein is aprecursor of the propeptide and further includes a signal peptide (e.g.amino acid sequence of SEQ ID NO: 2 or an amino acid sequence encoded bythe nucleotide sequence of SEQ ID NO: 1). It is the primary translationproduct. Also if the pre-propeptide is applied to a wound, it is cleavedto its active form preferably immediately before or during treatment ofsaid wound.

The term “having the ability to cleave fibrin and casein” preferablyonly refers to the activity of the mature protein but may also includethe pending activity (to be achieved by the mentioned cleavage(s)) ofthe pre-propeptide and the propeptide which are precursors of the matureprotein.

The term “protein” as used herein interchangeably with the term“polypeptide” or “peptide” describes linear molecular chains of aminoacids, including single chain proteins or their fragments, preferablycontaining more than 30 amino acids. An amino acid stretch of 30 aminoacids and less than 30 amino acids would normally only be called peptidebut not a polypeptide. Depending on the circumstances, the term“protein” or “polypeptide” herein may denote the mature protein (e.g.the amino acid sequence of SEQ ID NO: 4 or a amino acid sequence encodedby the nucleotide sequence of SEQ ID NO: 3), the propeptide (e.g. theamino acid sequence of SEQ ID NO: 6 or a amino acid sequence encoded bythe nucleotide sequence of SEQ ID NO: 5) or the pre-propeptide (e.g. theamino acid sequence of SEQ ID NO: 2 or a amino acid sequence encoded bythe nucleotide sequence of SEQ ID NO: 1). Polypeptides may further formoligomers consisting of at least two identical or different molecules.The corresponding higher order structures of such multimers are,correspondingly, termed homo- or heterodimers, homo- or heterotrimersetc. Furthermore, peptidomimetics of such proteins/polypeptides whereamino acid(s) and/or peptide bond(s) have been replaced by functionalanalogues are also encompassed by the invention. Such functionalanalogues include all known amino acids other than the 20 gene-encodedamino acids, such as selenocysteine. The terms “polypeptide”, “protein”and “peptide” also refer to naturally modified polypeptides/proteins andpeptides where the modification is effected e.g. by glycosylation,acetylation, phosphorylation and similar modifications which are wellknown in the art.

In connection with the fragments of the present invention, the term “thesame activity” refers to the biological activities as listed hereinwhich are present in fragments of the serine protease debrilaseaccording to the invention. Fragments according to this embodiment ofthe present invention can be polypeptides or peptides of equal or lessthan 30 amino acids, depending on their length.

In accordance with the present invention, the term “percent (%) sequenceidentity” describes the number of matches (“hits”) of identicalnucleotides/amino acids of two or more aligned nucleic acid or aminoacid sequences as compared to the number of nucleotides or amino acidresidues making up the overall length of the template nucleic acid oramino acid sequences. In other terms, using an alignment, for two ormore sequences or subsequences the percentage of amino acid residues ornucleotides that are the same (e.g. 80%, 85%, 90% or 95% identity) maybe determined, when the (sub)sequences are compared and aligned formaximum correspondence over a window of comparison, or over a designatedregion as measured using a sequence comparison algorithm as known in theart, or when manually aligned and visually inspected. This definitionalso applies to the complement of a test sequence.

Amino acid sequence analysis and alignment in connection with thepresent invention was carried out using the NCBI BLAST algorithm(Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, JinghuiZhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “GappedBLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402). The skilled person is alsoaware of suitable programs to align nucleic acid sequences.

Substitutions in the amino acid sequence of the serine protease of thepresent invention as compared to SEQ ID NOs: 2, 4 or 6 are preferablyconservative. This means that substitutions preferably take place withinone class of amino acids. For example, a positively charged amino acidis preferably mutated to another positively charged amino acid. The sameholds true for the classes of basic, aromatic or aliphatic amino acids.

The term “degenerate” in accordance with the present invention refers tothe degeneracy of the genetic code. Degeneracy results because a tripletcode designates 20 amino acids and a stop codon. Because four basesexist which are utilized to encode genetic information, triplet codonsare required to produce at least 21 different codes. The possible 4³possibilities for bases in triplets give 64 possible codons, meaningthat some degeneracy must exist. As a result, some amino acids areencoded by more than one triplet, i.e. by up to six. The degeneracymostly arises from alterations in the third position in a triplet. Thismeans that nucleic acid molecules having a different sequence than thatspecified above, but still encoding the same polypeptide lie within thescope of the present invention.

The problem often encountered when wounds do not heal in an appropriatetime is the removal of “fibrin cuffs” and necrotic tissue to enableinfiltration of fibroblasts and therefore the onset of the wound healingprocess. The present inventors have surprisingly found that the serineprotease encoded by the nucleic acid of the present invention hasexcellent properties to prepare the wound for natural healing bydissolving these fibrin cuffs. It is a biologically active serineprotease which is believed to be the major active principle of the woundhealing activity of Lucilia sericata. The enzyme is also referred toherein as “debrilase”, referring to its suggested use for thedebridement of wounds.

The larvae of Lucilia sericata are generally used in clinical practiceas living organisms to treat wounds which are resistant to conventionaltreatment. Since the maggot therapy is rather unpleasant to manypatients and the secretions of the insects are far from being a definedpharmaceutical composition the present inventors set out to identify atleast one of the active principles of wound debridement by a molecularapproach.

The generation of a cDNA-library from mRNA (the transcriptome) of secondinstar larvae fed on blood agar led to the identification of a nucleicacid encoding a trypsin-like protease and the corresponding amino acidsequence expressed with high abundance which is likely to be the majorprotease involved in wound debridement by larvae of Lucilia sericata.The enzyme was not found in cDNA libraries derived from mRNA isolatedfrom 5 days old larvae (third instar). This corresponds to the decreaseof debriding activity with increasing larval stages (Chambers et al.2003).

The screening of said libraries for proteolytic activity on caseinyielded several cDNA sequences encoding proteases in the transcriptomeof second instar larvae induced by blood-substrate. Analysis ofcDNA-sequences in the library of induced second instar larvae led to theidentification of proteases only present in the cDNA library generatedfrom second instar larvae but not in the transcriptome of larvae in thelater stage (third instar). Some of the proteases found exhibitednucleic acid sequence similarity to phosphochymotrypsin, trypsin orchymotrypsin, respectively. No metallo-, threonine-, cysteine-, asparticacid- or glutamic acid type proteases were found.

In a first agar plate screening on skim milk several cDNA clones werefound to secrete enzymes with caseinoloytic activity but only oneexhibited activity also on a fibrin containing substrate. This enzymewas characterised in detail by recombinant expression and assessment ofits biochemical properties in different assays.

Nucleic acid sequence analysis revealed three serine-type proteases andtwo trypsin-type proteases with high abundance within the mRNA ofinduced larvae. The trypsin-type proteases revealed fibrinolytic andcaseinolytic activity. Amino acid sequence analysis of one of thefibrinolytic proteases which corresponds to debrilase represented byamino acid sequence of SEQ ID NO. 4 and encoded by the nucleic acidsequence of SEQ ID NO: 2 showed a protein sequence identity of 76.1% toa trypsin-like protease from Sarcophaga sp. as closest similarity toknown enzymes using NCBI BLAST algorithm (Stephen F. Altschul, Thomas L.Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller,and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402). Debrilase was found to be expressed with high abundance,thus it is likely to be the major protease involved in wound debridementby larvae of Lucilia sericata. Debrilase is expressed and translated aspre-propeptide including a signal peptide and propeptidic amino acids(FIG. 5A). The activation of the enzymatic activity is accomplished bycleaving-off the signal peptide and by further cleaving the propeptidethereby generating the mature protein (SEQ ID NO. 4) of debrilase,wherein the propeptide is apparently further cleaved byauto-proteolysis. The mature protein is preferably encoded by thenucleic acid molecule consisting of SEQ ID NO. 3. The mature debrilasehas the ability to cleave fibrin and casein. Its activity can beinhibited completely by PMSF (phenylmethanesulfonyl fluoride) and APMSF(4-amidinophenylmethanesulfonyl fluoride). No inhibition was detectedusing inhibitors for metallo-, threonine-, cysteine- or aspartic acidtype proteases. This shows that the protein is a serine protease and hastrypsin-like activity.

It is known that progressive wound conditions encompass a wide pH range(Greener B et al 2005. Proteases and pH in chronic wounds. J Wound Care;14(2)). In another detailed study, 247 pH values in 39 patients withchronic wounds of varying origins were analysed over a period of 12months, detecting values from 5.45 to 8.65 (Dissemond, J. et al., 2003,pH-Wert des Milieus chronischer Wunden, Untersuchungen im Rahmen einermodernen Wundtherapie, Der Hautarzt 54, No. 12, 959-965). Debrilase ishighly stable and active in a pH range of 5-10 which makes it suitablein the application on chronic wounds exerting the wide pH range asdescribed above.

Moreover, in a cellular receptor activation assay debrilase was found tohave the ability to activate protease-activated receptor 2 (PAR 2). PAR2 is a member of the large family of 7-transmembrane receptors thatcouple to guanosine-nucleotide-binding proteins. PAR 2 is also a memberof the protease-activated receptor family. It is activated by trypsin,but not by thrombin. Trypsin accomplishes the proteolytic cleavage ofthe receptors extracellular amino terminus and the new amino terminusfunctions as a tethered ligand and activates the receptor. PAR 2 playsan important role in inflammation and pain, in fibroblast proliferation(Asano-Kato et al., Tryptase increases proliferative activity of humanconjunctival fibroblasts through protease-activated receptor-2, InvestOphthalmol Vis Sci., 2005 46(12):4622-6) and in formation of connectivetissue contributing to wound healing (Borensztajn K. et al., 2008,Factor Xa stimulates proinflammatory and profibrotic responses infibroblasts via protease-activated receptor-2 activation, 1: Am JPathol. 172(2):309-20). Thus, the trypsin-like enzyme of the inventionby cleaving the tethered ligand activates the receptor which thentriggers biochemical processes involved in promoting wound healing. Forexample, it is envisaged that debrilase can be utilised advantageouslyfor dissolving “fibrin cuffs” which are frequently observed onnon-healing wounds, for removing necrotic tissue and for triggering areceptor mediated cascade of immune and cellular responses.

This is the first description and characterization after recombinantexpression of a protease from Lucilia sericata with fibrinolytic,caseinolytic and PAR 2-activating activity and a wide pH-range of itsenzymatic activity on the molecular level. It is envisioned thatdebrilase and fragments thereof with debrilase activity have the abilityto prepare and pre-treat slow-healing or chronic wounds to make themsusceptible for natural healing processes and/or conventional medicaltreatment by debriding the wound, i.e. removal of dead tissue anddissolution of fibrin cuffs. Furthermore, cosmetic used are envisagedwhich involve skin treatments to improve skin appearance or texture.Application of the propetide to a wound will lead to a conversion to themature protein by enzymatic cleavage.

The present invention also relates to a vector comprising the nucleicacid molecule of the invention, i.e. a nucleic acid molecule encodingthe mature protein, the propeptide or the pre-propeptide as described inthe specification. In addition, the vector may contain a nucleic acidmolecule encoding a fragment as described above.

A vector according to this invention is capable of directing thereplication, and/or the expression of the nucleic acid molecule of theinvention and/or the expression of the polypeptide encoded thereby.

Preferably, the vector is a plasmid, cosmid, virus, bacteriophage oranother vector used conventionally e.g. in genetic engineering.

Exemplary plasmids and vectors are listed, for example, in Studier andcoworkers (Studier, W. F.; Rosenberg A. H.; Dunn J. J.; Dubendroff J.W., 1990. Use of the T7 RNA polymerase to direct expression of clonedgenes, Methods Enzymol. 185, 61-89) or the brochures supplied by thecompanies Novagen, Promega, New England Biolabs, Clontech and Gibco BRL.Other preferred plasmids and vectors can be found in: Glover, D. M.,1985, DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd.,Oxford; Rodriguez, R. L. and Denhardt, D. T. (eds), 1988, Vectors: asurvey of molecular cloning vectors and their uses, 179-204,Butterworth, Stoneham; Goedeel, D. V., 1990, Systems for heterologousgene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Russell, D.W., 2001, Molecular cloning: a laboratory manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, New York.

Non-limiting examples of suitable vectors include prokaryotic plasmidvectors, such as the pUC-series, pBluescript (Stratagene), thepET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen),lambda gt11, pJOE, the pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS,pTACT1 and vectors compatible with expression in mammalian cells likepREP (Invitrogen), pCEP4 (Invitrogen), pMClneo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo,pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectorpcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCOBRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech),pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pClNeo (Promega).Examples for plasmid vectors suitable for Pichia pastoris comprise e.g.the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

The nucleic acid molecule of the present invention referred to above mayalso be inserted into vectors such that a translational fusion withanother nucleic acid molecule is generated. The products arisingtherefrom are termed fusion proteins and will be described furtherbelow. The other nucleic acid molecules may encode a protein which maye.g. increase the solubility and/or facilitate the purification of theprotein encoded by the nucleic acid molecule of the invention.Non-limiting examples include pET32, pET41, pET43. The vectors may alsocontain an additional expressible nucleic acid coding for one or morechaperones to facilitate correct protein folding. Suitable bacterialexpression hosts comprise e.g. strains derived from BL21 (such asBL21(DE3), BL21(DE3) PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosettaa®.

Particularly preferred plasmids which can be used to introduce thenucleic acid encoding the serine protease of the invention into the hostcell are: pUC18/19 (Roche Biochemicals), pKK-177-3H (RocheBiochemicals), pBTac2 (Roche Biochemicals), pKK223-3 (Amersham PharmaciaBiotech), pKK-233-3 (Stratagene) and pET (Novagen). Further suitableplasmids are listed in PCT/EP03/07148. Very particular preference isgiven to an expression system which is based on plasmids belonging tothe pET series.

For vector modification techniques, see Sambrook and Russel, 2001.Generally, vectors can contain one or more origins of replication (ori)and inheritance systems for cloning or expression, one or more markersfor selection in the host, e.g., antibiotic resistance, and one or moreexpression cassettes. Suitable origins of replication include, forexample, the Col E1, the SV40 viral and the M 13 origins of replication.

The coding sequences inserted in the vector can e.g. be synthesized bystandard methods, or isolated from natural sources. Ligation of thecoding sequences to transcriptional regulatory elements and/or to otheramino acid encoding sequences can be carried out using establishedmethods. Transcriptional regulatory elements (parts of an expressioncassette) ensuring expression in prokaryotes or eukaryotic cells arewell known to those skilled in the art. These elements compriseregulatory sequences ensuring the initiation of the transcription (e.g.,translation initiation codon, promoters, enhancers, and/or insulators),internal ribosomal entry sites (IRES) (Owens et al., 2001) andoptionally poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers, and/ornaturally-associated or heterologous promoter regions. Preferably, thenucleic acid molecule of the invention is operably linked to suchexpression control sequences allowing expression in prokaryotes oreukaryotic cells. The vector may further comprise nucleotide sequencesencoding secretion signals as further regulatory elements. Suchsequences are well known to the person skilled in the art. Furthermore,depending on the expression system used, leader sequences capable ofdirecting the expressed polypeptide to a cellular compartment may beadded to the coding sequence of the nucleic acid molecule of theinvention. Such leader sequences are well known in the art. Specificallydesigned vectors allow the shuttling of DNA between different hosts,such as bacteria-fungal cells or bacteria-animal cells.

The nucleic acid molecules of the invention as described herein abovemay be designed for direct introduction or for introduction vialiposomes, phage vectors or viral vectors (e.g. adenoviral, retroviral)into the cell. Additionally, baculoviral systems or systems based onVaccinia Virus or Semliki Forest Virus can be used as vector ineukaryotic expression system for the nucleic acid molecules of theinvention. Expression vectors derived from viruses such as retroviruses,vaccinia virus, adeno-associated virus, herpes viruses, or bovinepapilloma virus, may be used for delivery of the nucleic acids or vectorinto targeted cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectors;see, for example, the techniques described in Sambrook, 2001 andAusubel, 2001.

Promoters which are particularly advantageous for implementing theinvention and which are to be used, in particular, in E. coli are knownto the skilled person (Sambrook, J.; Fritsch, E. F. and Maniatis, T.(1989), Molecular cloning: a laboratory manual, 2nd ed., Cold SpringHarbor Laboratory Press, New York). Further suitable promoters are thoseselected from T7, lac, tac, trp, ara or rhamnose-inducible promoters.Other promoters are mentioned in (Cantrell, S A (2003) Vectors for theexpression of recombinant proteins in E. coli. Methods in Molecularbiology 235: 257-275; Sawers, G; Jarsch, M (1996) Alternative principlesfor the production of recombinant proteins in Escherichia coli. AppliedMicrobiology and Biotechnology 46(1): 1-9). Very particular preferenceis given to using the T7 promoter in the vector according to theinvention (Studier, W. F.; Rosenberg A. H.; Dunn J. J.; Dubendroff J.W.; (1990), Use of the T7 RNA polymerase to direct expression of clonedgenes, Methods Enzymol. 185, 61-89; or brochures supplied by thecompanies Novagen or Promega).

Examples for regulatory elements permitting expression in eukaryotichost cells are the AOX1 or GAL1 promoter in yeast or the CMV-(Cytomegalovirus), SV40-, RSV-promoter (Rous sarcoma virus), chickenbeta-actin promoter, CAG-promoter (a combination of chicken beta-actinpromoter and cytomegalovirus immediate-early enhancer), the gai10promoter, human elongation factor 1α-promoter, CMV enhancer, CaM-kinasepromoter, the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) polyhedral promoter or a globin intron in mammalian and otheranimal cells. Besides elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site or the SV40, lacZ and AcMNPV polyhedral polyadenylationsignals, downstream of the nucleic acid.

The co-transfection with a selectable marker such as kanamycin orampicillin resistance genes for culturing in E. coli and other bacteriaallows the identification and isolation of the transfected cells.

Selectable markers for mammalian cell culture are the dhfr, gpt,neomycin, hygromycin resistance genes. The transfected nucleic acid canalso be amplified to express large amounts of the encoded (poly)peptide.The DHFR (dihydrofolate reductase) marker is useful to develop celllines that carry several hundred or even several thousand copies of thegene of interest. Another useful selection marker is the enzymeglutamine synthase (GS) (Murphy et al. 1991; Bebbington et al. 1992).

Using such markers, the cells are grown in selective medium and thecells with the highest resistance are selected.

In a further embodiment, the present invention relates to a host celltransformed, transduced or transfected with the vector of the invention.

Host cells into which vectors containing the nucleic acid molecule ofthe invention can be cloned are used for replicating and isolating asufficient quantity of the recombinant enzyme. The methods used for thispurpose are well known to the skilled person (Sambrook, J.; Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nded., Cold Spring Harbor Laboratory Press, New York).

Suitable prokaryotic host cells comprise e.g. bacteria of the speciesEscherichia, such as strains derived from E. coli BL21 (e.g. BL21(DE3),BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE, BL21 codon plus, BL21(DE3)codon plus), Rosetta®, XL1 Blue, NM522, JM101, JM109, JM105, RR1, DH5α,TOP 10, HB101 or MM294. Further suitable bacterial host cells areStreptomyces, Salmonella or Bacillus such as Bacillus subtilis. E. colistrains are preferred prokaryotic host cells in connection with thepresent invention.

Suitable eukaryotic host cells are e.g. yeasts such as Saccharomycescerevisiae, Hansenula polymorpha or Pichia sp. such as P. pastoris,insect cells such as Drosophila S2 or Spodoptera Sf9 cells, or plantcells.

Very particular preference is given to an expression system which ispresent in E. coli BL21 as a procaryotic host and Pichia pastoris as aeucaryotic host.

Mammalian host cells that could be used include human Hela, HEK293, H9and Jurkat cells, mouse NIH3T3 and C127 cells, COS1, COS 7 and CV1,quail QC1-3 cells, mouse L cells. Bowes melanoma cells and Chinesehamster ovary (CHO) cells.

The present invention furthermore relates to a method of producing aserine protease, a fragment, a propeptide or pre-propeptide thereof asdescribed above comprising, culturing the host cell of the invention andisolating the serine protease, the fragment, the propeptide or thepre-propeptide produced.

Suitable conditions for culturing a prokaryotic or eukaryotic host arewell known to the person skilled in the art. For example, suitableconditions for culturing bacteria are growing them under aeration inLuria Bertani (LB) medium. To increase the yield and the solubility ofthe expression product, the medium can be buffered or supplemented withsuitable additives known to enhance or facilitate both. E. coli can becultured from 4 to about 37° C., the exact temperature or sequence oftemperatures depends on the molecule to be overexpressed. In general,the skilled person is also aware that these conditions may have to beadapted to the needs of the host and the requirements of the polypeptideexpressed. In case an inducible promoter controls the nucleic acid ofthe invention in the vector present in the host cell, expression of thepolypeptide can be induced by addition of an appropriate inducing agent.Suitable expression protocols and strategies are known to the skilledperson.

Depending on the cell type and its specific requirements, mammalian cellculture can e.g. be carried out in RPMI or DMEM medium containing 10%(v/v) FCS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycine. Thecells can be kept at 37° C. in a 5% CO₂, water saturated atmosphere.

Suitable media for insect cell culture is e.g. TNM+10% FCS or SF900medium. Insect cells are usually grown at 27° C. as adhesion orsuspension culture. Suitable expression protocols for eukaryotic cellsare well known to the skilled person and can be retrieved e.g. from inSambrook, 2001.

Methods of isolation of the polypeptide produced are well-known in theart and comprise without limitation method steps such as ion exchangechromatography, gel filtration chromatography (size exclusionchromatography), affinity chromatography, high pressure liquidchromatography (HPLC), reversed phase HPLC, disc gel electrophoresis orimmunoprecipitation, see, for example, in Sambrook, 2001.

The present invention further relates to a serine protease, fragment,propeptide, or pre-propeptide encoded by the nucleic acid molecule ofthe invention or produced by the method of the invention. As mentioned,this serine protease is also referred to as “debrilase”.

As described above, the serine protease of the invention ischaracterized in that it (i) originates from Lucilia sericata, (ii)exhibits proteolytic activity against fibrin, casein andTosyl-Gly-Pro-Arg-AMC, but not against Suc-Ala-Ala-Phe-AMC. (iii) Theproteolytic activity of the serine protease of the invention againstcasein and fibrin is inhibited by the serine proteinase inhibitors PMSFand APMSF and (iv), debrilase preferably activates theprotease-activated receptor PAR-2. It could surprisingly be shown thatit is active in a wide pH range between pH 5 and 10 (see FIG. 3B).

As mentioned, the invention further relates to a propeptide of theserine protease of the invention which is cleaved to its active formpreferably immediately before or during treatment of a wound. Thispropeptide preferably has the amino acid sequence of SEQ ID NO: 6 andalso preferably is encoded by the nucleic acid sequence if SEQ ID NO: 5.

In a further embodiment, the present invention relates to a fusionprotein comprising the serine protease of the invention, the fragment ofthe serine protease of the invention, the propeptide of the invention orthe pre-propeptide of the invention.

In addition to the amino acid sequence of the serine protease(debrilase), the fragment, the propeptide or the pre-propeptide thereofof the present invention debrilase, a fusion protein according to thepresent invention contains at least one additional, heterologous aminoacid sequence. Often, but not necessarily, these additional sequenceswill be located at the N- or C-terminal end of the polypeptide. It maye.g. be convenient to initially express the polypeptide as a fusionprotein from which the additional amino acid residues can be removed,e.g. by a proteinase capable of specifically trimming the polypeptide ofthe present invention.

Exemplary fusion proteins of debrilase, a fragment thereof or the (pre-)propeptide of debrilase further comprise a peptide or protein which canfunction to mediate the adhesion of the fusion protein to a matrixcontained in wound dressing in order to achieve a stable incorporationof debrilase into the medical article of the invention. The fusionprotein can be selected from the group of surface active proteins orantibodies and fragments thereof. The properties of antibodies andfragments thereof will be described further below in connection with theantibody of the invention.

The present invention furthermore relates to a composition comprisingthe serine protease of the invention, the fragment of the serineprotease or fragment thereof of the invention, the propeptide of theinvention, the pre-propeptide of the invention, the fusion protein ofthe invention, the nucleic acid of the invention, the vector of theinvention, the host cell of the invention, or combinations thereof.

In a preferred embodiment, the composition is a pharmaceuticalcomposition.

In accordance with the present invention, the term “pharmaceuticalcomposition” relates to a composition for administration to a patient,preferably a human patient. The pharmaceutical composition is preferablyused in an adjuvant situation, i.e. for preparing the slow healing woundfor natural healing processes or conventional medical treatment byremoving necrotic tissue and dissolving fibrin cuffs (debridement). Thepharmaceutical composition of the invention comprises the compoundsrecited above, alone or in combination. The composition may be in solidor liquid form and may be, inter alia, in the form of (a) powder(s), (a)solution(s) or (an) aerosol(s), cream(s), ointment(s) or gel(s). Thepharmaceutical composition of the present invention may, optionally andadditionally, comprise a pharmaceutically acceptable carrier. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Examples of suitable pharmaceuticalcarriers are well known in the art. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thepharmaceutical composition can be administered topically. The dosageregimen corresponding to a suitable dose for administration will bedetermined by the attending physician and clinical factors which may,inter alia, depend on the size of the wound, the stage or severity ofits condition. The concentration of the compound(s) as recited above ina composition for topical application can be in the range of 0.001 to 1%(w/w), preferably 0.01-0.1% (w/w). Topical application on wounds ispreferably repeated in one or more than one daily applications.

The compound(s) as recited above can be used as active ingredient inpharmaceutical composition for the debridement of chronic andslow-healing wounds by dissolution of the fibrin cuff. Thepharmaceutical composition according to the invention is preferably usedin an adjuvant situation, i.e. for preparing the slow healing wound fornatural healing processes or conventional medical treatment by removingnecrotic tissue and dissolving fibrin cuffs (debridement).

The pharmaceutical composition of the invention can be applied incombination with (solid) carriers or matrices such as dressing(s), bandaid(s) or tape(s). The compound(s) can be covalently or non-covalentlybound to said carrier or matrix. These can be applied to wounds topromote wound healing.

For example, the compound(s) may be incorporated into a dressing to beapplied over the wound. Examples of such dressings include staged orlayered dressings incorporating slow-release hydrocolloid particlescontaining the wound healing material or sponges containing the woundhealing material optionally covered by conventional dressings. Theconcentration of a solution of the pharmaceutical composition to beimmobilised in a matrix of a wound dressing is generally in the range of0.001 to 1% (w/v) preferably 0.01-0.1% (w/v). Furthermore, thecompound(s) as recited above can be incorporated into a suitablematerial capable of delivering the enzyme to a wound in a slow releaseor controlled release manner.

Renewal of the wound dressing in appropriate intervals (e.g. 24 h)should be repeated until complete removal of fibrin cuff and necrotictissue is accomplished. The dressing is suitable for any woundcomprising necrotic tissue including leg ulcers, pressure sores,diabetic foot ulcers and burns. Preferably, the dressing comprises alayer of absorbent material such as hydrocolloids, foam, e.g.polyurethane foam, alginates, chitosan, so it may be used on exudatingwounds whilst protecting the surrounding skin from maceration. Thewound-contacting layer can be coated with an excipient, e.g. petrolatum,to prevent the dressing from sticking to the wound. The compound(s)according to the invention may be incorporated in a polymeric materialsuch as e.g. cellulose, polylactates, polyvinyls, acrylic copolymers andmay be integrated in the dressing in different ways. In one embodimentthe polymeric material is in the form of a film on the wound-facingsurface of the dressing. A high concentration of the compound(s) isdesired on the surface of the dressing contacting the wound bed in orderto obtain a more effective debridement due to a high initial release ofthe enzyme. The film may be in the form of a layer or it may be coatedon a net.

Especially in the case of third degree burns, band aids comprisingdebrilase as debriding enzyme can be used in a wound debridementtherapy.

In a formulation comprising the above compound(s), optionally comprisedin a sterile carrier, the pharmaceutical composition of the invention inliquid form can be sprinkled over the wound area or, in solid or inliquid form, be incorporated into a carrier to be applied to the wound.

In a preferred embodiment, the serine protease of the invention iscomprised in a solid carrier such as a wound dressing or tape in itsinactive form and may be activated through cleavage of a(pre-)propeptide as described above by different means e.g. when broughtin contact with wound exudate. It is preferred that the enzyme activitylasts during the recommended wear time of the dressing, depending on theamount of exudate.

A gel formulation of the pharmaceutical composition of the presentinvention further comprises at least one gel forming agent commonly usedin pharmaceutical gel formulations. Examples of gel forming agents arecellulose derivatives such as methyl cellulose, hydroxyethyl cellulose,and carboxymethyl cellulose; vinyl polymers such as polyvinyl alcohols,polyvinyl pyrrolidones; and carboxypoly-methylene derivatives such ascarbopol. Further gelling agents that can be used for the presentinvention are pectins, gums, alginates, agar and gelatine. Furthermore,the gel or emugel formulation may contain auxiliary agents commonly usedin this kind of formulations such as preservatives, antioxidants,stabilizers, colorants and perfumes.

In another preferred embodiment, the composition is a cosmeticcomposition.

A cosmetic composition according to the invention is for use innon-therapeutic applications.

Cosmetic compositions may also be defined by their intended use, ascompositions intended to be rubbed, poured, sprinkled, or sprayed on, orotherwise applied to the human body for cleansing, beautifying,promoting attractiveness, or altering the appearance.

The particular formulation of the cosmetic composition according to theinvention is not limited. Envisaged formulations include rinsesolutions, emulsions, creams, milks, gels such as hydrogels, ointments,suspensions, dispersions, powders, solid sticks, foams, sprays andshampoos. For this purpose, the cosmetic composition according to theinvention may further comprise cosmetically acceptable diluents and/orcarriers. Choosing appropriate carriers and diluents in dependency ofthe desired formulation is within the skills of the skilled person.Suitable cosmetically acceptable diluents and carriers are well known inthe art and include agents referred to in Bushell et al. (WO2006/053613). Preferred formulations for said cosmetic composition arerinse solutions and creams.

The application of the composition of the invention in cosmetics isaiming at treating the skin enzymatically for peeling and smootheningand/or intervention with scar formation. A suitable concentration of thecompound(s) of the invention for cosmetic use is believed to be in therange of 0.0001 to 1% (w/v), preferably 0.0001 to 0, 1% (w/v), even morepreferably 0.001 to 0.1% (w/v).

Preferred amounts of the cosmetic compositions according to theinvention to be applied in a single application are between 0.1 and 10g, more preferred between 0.1 and 1 g, most preferred 0.5 g. The amountto be applied also depends on the size of the area to be treated and hasto be adapted thereto.

In another aspect, the present invention relates to the serine proteaseof the invention, the fragment of the serine protease of the invention,the propeptide of the invention, the pre-propeptide of the invention,the fusion protein of the invention, the nucleic acid of the invention,the vector of the invention or the host cell of the invention for thepreparation of a cosmetic composition for skin peeling, skin smootheningor the intervention with scar formation, most preferably in the form ofan adjuvant therapy. The invention furthermore relates to the serineprotease of the invention, the fragment of the serine protease of theinvention, the propeptide of the invention, the pre-propeptide of theinvention, the fusion protein of the invention, the nucleic acid of theinvention, the vector of the invention or the host cell of the inventionfor use in the treatment of wounds.

The particulars of the composition comprising the compound(s) of theinvention to be used in connection with the above applications incosmetics and therapeutics have been described above.

In a preferred embodiment, the wounds are chronic or slow healingwounds.

In a further aspect, the invention relates to a method for treating awound to promote healing thereof in a human or non-human mammal whichcomprises applying to the wound a therapeutically effective amount of asterile composition comprising the serine protease, fragment thereof,the propeptide, the pre-propeptide, the fusion protein, the nucleicacid, the vector or the host cell according to the invention as activeingredient. The invention also relates to the use of a compound of theinvention as recited above in the preparation or manufacture of amedical article like a band aid, dressing or tape for the debridement ofwounds and dissolution of the fibrin cuff.

The invention furthermore relates to the serine protease of theinvention, the fragment of the serine protease of the invention, thepropeptide of the invention, the pre-propeptide of the invention, thefusion protein of the invention, the nucleic acid of the invention, thevector of the invention or the host cell of the invention for use in thetreatment of skin diseases accompanied by skin lesions and/or impairedwound healing. Examples of such skin lesions and/or impaired healing arepsoriasis, atopical dermatitis, contact dermatitis and eczema andurticaria.

In a preferred embodiment, the composition of the invention additionallycomprises at least one component selected from the group of a furtherprotease, nuclease, excipient, anti-microbial agent and pain-relievingagent.

The debridement process as well as dressing changes may give rise topain for the patient, and thus it may be preferred to incorporate apain-relieving agent such as an analgesic or an anaesthetic compound inthe dressing of the present invention.

The dressing of the present invention may further comprise debridingcompositions other than enzymes, which may have additive or synergisticdebriding effect. An example of such a compound may be urea.

The invention also relates to an antibody or fragment or derivativethereof specifically binding to the serine protease of the invention, ora fragment thereof or the propeptide or pre-propeptide of the serineprotease of the invention.

An antibody according to the present invention specifically binds to theserine protease (debrilase) as described in detail herein above orfragments thereof having the activity of debrilase as described above orthe propeptide or pre-propeptide of the serine protease of the presentinvention.

The antibody of the present invention can be, for example, polyclonal ormonoclonal. The term “antibody” also comprises derivatives or fragmentsthereof which still retain the binding specificity. Such fragmentscomprise, inter alia, Fab fragments, F(ab′)₂, Fv fragments or scFvderivatives. Techniques for the production of antibodies and fragmentsthereof are well known in the art and described, e.g. in Harlow and Lane“Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press,1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” ColdSpring Harbor Laboratory Press, 1998. These antibodies can be used, forexample, for immunoprecipitation or affinity purification of debrilaseor fragments thereof.

The antibody of the invention also includes embodiments such aschimeric, single chain and humanized antibodies. Various procedures areknown in the art and may be used for the production of such antibodiesand/or fragments. Further, techniques described for the production ofsingle chain antibodies can be adapted to produce single chainantibodies specific for debrilase or fragments thereof etc. describedabove. Also, transgenic animals may be used to express humanizedantibodies specific for debrilase or fragments thereof. Most preferably,the antibody of this invention is a monoclonal antibody. For thepreparation of monoclonal antibodies, any technique that providesantibodies produced by continuous cell line cultures can be used.Examples for such techniques include the hybridoma technique (Köhler andMilstein Nature 256 (1975), 495-497), the trioma technique, the humanB-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985), 77-96). Surface plasmon resonance as employed in the BIAcoresystem can be used to increase the efficiency of phage antibodies whichbind to an epitope of the compounds described above (Schier, HumanAntibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods183 (1995), 7-13). It is also envisaged in the context of this inventionthat the term “antibody” comprises antibody constructs that may beexpressed in cells, e.g. antibody constructs which may be transfectedand/or transduced via, amongst others, viruses or plasmid vectors. Oncethe antibody according to the present invention has been obtained, theantibody itself or the DNA encoding it can be sequenced providing forthe information to recombinantly produce the antibody of the inventionin small or large scale. Methods of the production of a recombinantantibody are known to the person skilled in the art.

The term “specifically binds”, interchangeably used with “specificallyinteracts with”, in accordance with the present invention means that theantibody does not or essentially does not cross-react with an epitope ofsimilar structure. Cross-reactivity of a panel of antibodies underinvestigation may be tested, for example, by assessing binding of saidpanel of antibodies under conventional conditions to the epitope ofinterest as well as to a number of more or less (structurally and/orfunctionally) closely related epitopes. Only those antibodies that bindto the epitope of interest in its relevant context (e.g. a specificmotif in the structure of a protein) but do not or do not essentiallybind to any of the other epitopes are considered specific for theepitope of interest and thus to be antibodies in accordance with thisinvention. Corresponding methods are described e.g. in Harlow and Lane,1988 and 1999, loc cit.

The invention is herein described, by way of example only, withreference to the accompanying drawings for purposes of illustrativedescription of the preferred embodiments of the present invention.

The figures show:

FIG. 1: Diffusion assay on agar containing casein showing a clearingzone around a debrilase secreting clone of E. coli.

FIG. 2: Activity assay in agar containing fibrin showing a clearing zonearound debrilase active sample A) debrilase positive clone growing onfibrin containing substrate, B) agar diffusion assay with crude extractsfrom a debrilase-positive recombinant E. coli clone: (a) undiluted crudeextract b) 1:10 dilution in PBS c) 1:50 dilution in PBS.

FIG. 3: A) enzyme assay for identifying the activity profile ofrecombinant debrilase by use of known serine protease (PMSF) and trypsinlike serine protease (APMSF) inhibitors; B) activity assay withdebrilase in puffers of different pH-values to identify the pH-profileof recombinant debrilase.

FIG. 4: Activation of PAR 2 in a cell based receptor assay with HEK232cells with Ca²⁺-flux as read-out.

FIG. 5: Alignment of the pre-propeptide amino acid of debrilase (A)(SEQ. ID. NO:2) and cDNA nucleotide sequence (B) (SEQ. ID. NO:1) withthe nearest neighbour sequence being the trypsin like protein fromSarcophaga bullata (SEQ. ID. NOs:9 and 10) showing 76% identity fornucleotide and protein sequence using NCBI BLAST algorithm. (A) Thesignal peptide of debrilase consists of amino acids 1 to 16 (the signalpeptide is shown bold), the propeptide consists of amino acids 17 to 254(propeptidic amino acids 17 to 26 are underlined) and theproteolitically processed mature polypeptide of debrilase consists ofamino acids 27 to 254.

FIG. 6: (A) SDS PAGE of culture supernatant: 1) after centrifugation; 2)pre-filtration [0.45 μm]; 3) sterile filtration [0.22 μm], MW of thepropeptide 25.7 kDa. (B) Visualisation of activated mature Debrilase; 5)after dialysis and subsequent pH shift from 5.5 to 8; 6) affinitychromatography column flow-through (unbound protein); 7) wash flowthrough, (8 to 12) fractionated elution 1 to 5, respectively (molecularweight of the mature protein 24.6 kDa).

The examples illustrate the invention.

EXAMPLES General Methods and Materials

Isolation of mRNA of Different Larval Stases from Lucilia sericata

2 days old maggots from the green bottlefly (Lucilia sericata) werepurchased from BioMonde (BioMonde, 22885 Barsbüttel, Germany). Isolationof total RNA was done after feeding for 24 hours (second instar stage)on blood-agar (1.5% (w/v) agar containing 10% bovine blood) to enhanceproduction of proteases and after 3 days when the production of enzymesis markedly decreased in the third instar stage (Chambers et al. 2000,Degradation of extracellular matrix components by defined proteinasesfrom the greenbottle larva Lucilia sericata used for the clinicaldebridement of non-healing wounds, Brit J Dermatol. 148: 14-23). Forisolation of total-RNA 3 larvae (ca. 30 mg) were shock frozen withliquid nitrogen and pulverised mechanically. RNA was isolated using thekit “Total RNA Isolation, NucleoSpin RNA II” (purchased fromMacherey-Nagel, 52313 Duren, Germany) yielding 47 μg total RNA.

Generation of an E. coli cDNA Library from Total RNA Isolated fromLarvae of Lucilia sericata

The generation of a cDNA-library from said Lucilia sericata RNA wasaccomplished according to the “SMART cDNA Library Construction Kit UserManual” (CLONTECH Laboratories, Inc.). In vitro packaging of the finalligation reaction and subsequent transformation in E. coli XL-1 Blueresulted in 1.5×10E6 primary plaque forming units.

Primary phages were harvested and stored in phage stabilization buffercontaining 7% DMSO at −80° C. for subsequent infection and mass excisionin E. coli.

Conversion of the lambda phage library into the corresponding plasmidlibrary was performed by “Cre” recombinase-mediated mass excision of thephage embedded plasmids according to the “SMART cDNA LibraryConstruction Kit User Manual” (CLONTECH Laboratories, Inc.).

Heterogeneity and Quality of the Library was Tested by RestrictionAnalysis

Identification of Proteases from Lydiai Sericata cDNA-Library

Colony forming units resulted from mass excision described above werescreened on agar media containing 2% skim milk under selectiveconditions. Expression of heterologous proteases was driven by thevector comprising inducible P_(lac) promoter. Colonies expressingheterologous proteases were detected by the formation of clearing zonesin the turbid Skim Milk medium around the colonies.

Isolation and Purification of Debrilase Protein from E. Coli CultureExpression Culture

In order to obtain enzyme samples of the debrilase containing sufficientenzymatic activity for a characterisation of the enzyme either the cDNAclone expressing the corresponding protease or a more suited expressionconstruct set up in a typical expression vector like e.g thepET26b-vector (Novagen) and a suitable expression host like e.g. E. coliRosetta (DE3) (Novagen) were used. For the construction of theexpression constructs, the corresponding debrilase genes were PCRamplified to introduce unique restriction enzyme recognition sequencesupstream and downstream of the open reading frame (ORF) which allowed toligate the genes encoding the protease with the expression vector e.g.pET26b in a definite way. The restriction enzyme recognition sequenceswere chosen on the basis of their absence in the coding region of thedebrilase gene and could be e.g. NdeI, HindIII, EcoRI, XhoI. The absenceof unwanted second site mutations due to erroneous amplification by thepolymerase was confirmed by sequencing of the cloned amplicon.

The cDNA clones or the expression constructs were used to inoculate e.g.200 ml of culture medium complemented with the appropriate antibiotic ina 1 l Erlenmayer flask. LB-medium and antibiotics in the followingconcentration were used: 100 μg/ml ampicillin, 25 μg/ml kanamycin,chloroamphenicol 12.5 μg/ml. The initial optical density (OD₅₈₀) wasadjusted to 0.05 and the cells grown at a temperature of 28° C. on agyratory shaker. When the optical density reached the value of about 1the expression from the lac-promoter of pTriplEx2 (Clontech) or from theT7-promoter of vectors from the pET-vector series e.g. pET26 was inducedby addition of IPTG in the concentration of 20 μM-500 μM. Cells wereharvested 4 to 20 h after induction by centrifugation. The cell sedimentwas resuspended in 5 ml 1×PBS, pH 7.0 and the cells disrupted byultrasonication.

The molecular weight of debrilase is 27.4 kD for the complete proteinincluding signal sequence, 25.7 for the hypothetical pro-peptide and24.6 for the mature protein.

Solid Phase Activity Assay for Fibrinolytic Activity

To identify fibrinolytic activity in extracts of casein degrading clonesa solid agar assay was used. For this, a solution A containing 200 mgagarose, 87 mg NaCl and 3 mg CaCl₂ were added to 10 ml 0.1 M Tris pH7.4and incubated at 50° C. To a second solution B containing 10 ml 0.9%NaCl and 100 mg Fibrin (Sigma F3879), 20 μl Plasminogen (2 units/mg,Sigma P7397) e added and pre-incubated at 50° C. Both solutions werecombined with 12 μl Thrombin (175-350 NIH units, Sigma T4265) and spreadon agar plates using micro-well forming devices. In case of afibrinolytic activity a clear zone appears in the opaque medium aroundextract containing cavities.

To identify fibrinolytic activity in casein degrading clones a solidagar assay was used. Therefore a medium A consisting of 10 ml 2× LuriaBertani containing 200 mg agarose, 87 mg NaCl and 3 mg CaCl₂ wasincubated at 50° C. To a solution B containing 10 ml 0.9% NaCl and 100mg Fibrin (Sigma F3879), 20 μl plasminogen (2 units/mg, Sigma P7397) wasadded and pre-incubated at 50° C. Both solutions were combined with 12μl thrombin (175-350 NIH units, Sigma T4265) and appropriate antibioticsfor selection and poured on agar plates under sterile conditions forminga opaque agar medium. In case of fibrinolytic activity expressed byheterologous clones a clear zone appears around the colony after overnight incubation.

Liquid Phase Activity Assay and Inhibitory Profiling

Quantification of proteolytic activity was conducted by a fluorescenceassay in 96-well plates with BODIPY-FL-Casein (Molecular Probes) used assubstrate. Hereby, on the undigested substrate casein the labellingmolecules are in close contact to each other and therefore fluorescenceis suppressed by a quenching mechanism. In case of hydrolysis thesemolecules separate from each other and fluorescence can be excited at485 nm and measured at 520 nm.

A standard assay contained 5 μg/ml BODIPY-FL-Casein in 100 μl PBS bufferand a series of dilutions of protease extract. Samples were incubatedfor 60 min at 37° C. and fluorescence was measured using aspectrophotometer (NovoStar, BMG LABTECH, Offenburg, Germany), applyingthe following parameters: excitation 485 nm, emission 520 nm, gain 5,one cycle, 10 flashes. Serine protease inhibitors APMSF(4-amidinophenylmethanesulfonyl fluoride) and PMSF(phenylmethanesulfonyl fluoride) were used for testing the specificityof the isolated protease. The following assay concentration were used:

PMSF = serine protease inhibitor 5 mmol/l APMSF = trypsin like serineprotease inhibitor 1 mmol/l

The Sequence Listing submitted herewith electronically is incorporatedherein by reference.

The following examples are illustrative, but not limiting the scope ofthe present invention. Reasonable variations, such as those occur to theperson skilled in the art, can be made herein without departing from thescope of the present invention.

Pichia pastoris Expression Culture

Heterologous expression of the serine protease of the invention, i.e.debrilase was carried out in the methylotrophic yeast Pichia pastoris,which is classified as a GRAS-organism by the Food and DrugAdministration and is therefore established for pharmaceuticalproduction processes. For expression an integrative vector system wasused, providing a methanol inducible promoter. The native signal peptideof debrilase (“MFRFVALFAFVSCALA”) (SEQ. ID. NO:7) was substituted by thevector encoded-factor signal sequence from Saccharomyces cerevisiae tofacilitate efficient secretion in Pichia.

Fermentation was carried out in mineral medium with glycerol as solecarbon source in the first batch phase, fed batch mode was started afterconsumption of initial batch glycerol. Induction of heterologousexpression was initiated by supplementation of methanol asinducer/carbon source and sorbitol as additional non-inhibiting feedduring gene expression.

Stationary growth of the culture was reached after batch/fed-batch (FB)phase leading to an Optical Density of about 600. Induction wasinitiated by adding MeOH. To prevent heterologous enzyme from beingactivated autocatalytically at this point, process temperature wasdecreased from 30° C. to 20° C. Expression of debrilase was supported inthe end phase of the process by feeding. Autocatalytic activation of thepropeptide was achieved by increasing the pH of the culture medium from5.5 to 6.8, during the purification procedure (see below).

Purification of Debrilase Protein from Pichia pastoris CultureExpression Culture

Purification was carried out by affinity chromatography using serineprotease inhibitor benzamidine coupled to sepharose carrier material (GEHealthcare). After removal of unbound protein purified the serineprotease of the invention was harvested by competitive elution usingbuffer with addition of free benzamidine. SDS PAGE of the downstreamprocedure is visualized in FIG. 6. After subsequent dialysis againstcitrate storage buffer the purified protein was lyophilized anddisplayed as white dry powder.

N-Terminal Sequencing of Recombinantly Expressed Debrilase

For Edman sequencing the blot was fitted into the sample preparationcartridge. For determination of the amino acid sequence the proteinsequencer Procise 492 (Applied Biosystems) was used. Reagents andprotocols were applied as advised by the manufacturer. The resultingchromatograms were analysed using appropriate software (AppliedBiosystems). Prior to each sample a standard sample and a blank wererun.

Example 1 Screening for Proteases in an Expression Library Generatedfrom Lucilia sericata

A phage library of 8×10E6 primary clones was screened for the expressionof proteases. For this end, the phage library was transferred into aplasmid library by co-infection of E. coli with a f1 type helper phageaccording to the “SMART cDNA Library Construction Kit User Manual”(CLONTECH Laboratories, Inc.). The resulting colonies harbour theintrinsic plasmids excised from the phage vector.

The heterogeneity of the plasmid library was tested by isolation offorty plasmids from representative clones. In fact, every plasmidharboured an insert and these inserts showed complete diversity in size.In total, 3×10E5 cfu were screened on solid media containing 2% skimmilk under selective conditions.

By isolation and sequencing of plasmid DNA from sixteen of thesehalo-forming colonies a pre-propeptide sequence of a protease with 76%identity on the amino acid level to a trypsin-like enzyme fromSarcophaga bullata was identified (SEQ ID NO: 2).

Example 2 Characterisation of a Fibrinolytic Protease

The identification of fibrinolytic activity was performed in agar plateassays described above. Colonies expressing fibrinolytic activity wereidentified by streaking out recombinant cells on nutrient agarcontaining turbid fibrin substrate. Cells harbouring plasmids with theidentified protease showed clear zones around the colony after overnight incubation at 37° C.

Fibrinolytic activity in cell free crude extracts was determined usingbuffered agar medium containing fibrin. Plates contained wells with acapacity of up to 200 μl which were formed using microplate devicesduring solification of the agar medium. Recombinant cells were sonicatedand the resulting extract was centrifuged, places into the agar wellsand incubated over night at 37° C. Crude extract with fibrinolyticactivity was detected by a clear zone around microwells of therecombinant clone harbouring the protease gene of SEQ ID NO. 1.

Example 3 Profiling of a Fibrinolytic Protease

The recombinant protease type was identified in a liquid assay in a96-well plate using inhibitors specific for different types ofproteases. Herein, specific inhibitors for trypsin(4-amidinophenylmethyl-sulphonyl fluoride, APMSF), serine(phenylmethylsulphonyl fluoride, PMSF), aspartyl-(pepstatin A) andmetallo-type proteases (1,10-phenanthroline) were measured. As shown inFIG. 3 A, the inhibitor-profile of the recombinant clone suggests atrypsin type activity, which corresponds to comparative amino acidsequence alignment data of SEQ ID NO: 2 with the BLAST data base,showing the closest homology being 76% to an enzyme from Sarcophagabullata (FIG. 5).

Example 4 Stability at Different pH-Values

Activity of the debrilase was measured in liquid assay using appropriatebuffer systems in the range of 3-10 (0.1 to 0.2 M citric acid buffer, pH3-7; 0.05 M tris buffer, pH 8; 0.1 M carbonate buffer, pH 9-10) and thefluorogenic substrate Z-Gly-Gly-Arg-AMC. The release of7-amino-4-methylcoumarin (AMC) was measured with a BMC NovostarFluorometer (λ_(excitation) 365 nm, λ_(emission) 440 nm). The resultsare shown in FIG. 3 B. Debrilase exhibits enzymatic activity in apH-range of 5-10 which corresponds to the similarly wide pH-range of thewound milieu.

Example 5 Activation of PAR 2 (Calcium Imaging)

The activity of debrilase as agonist of PAR 2 was monitored over time ina cell based fluorescence assay by use of a wild type HEK293 cell lineendogenously expressing human PAR2. In brief, 1 day prior to performingthe assay, HEK293 cells expressing human PAR2 were plated onto 96-well,black-walled, assay plates, at a density of 45,000 cells per well. Usinga 96-well microplate reader (FlexStation®, Molecular Devices, Sunnyvale,Calif.), the change of the cellular calcium concentration was monitoredby use of the calcium sensitive fluorescent dye fluo-4 (excitation 494nm, emission 516 nm). The PAR 2 agonist trypsin (8 nM) was used aspositive control. The dye-loaded cells in plates were placed into thefluorescence microplate reader to monitor fluorescence (excitation 488nm, emission 520 nm) change after the addition of 50 μl assay buffer(118 mM NaCl; 4.7 mM KCl; 1.2 mM MgSO₄; 1.2 mM KH₂PO₄ 4.2 mM NaHCO₂; 1.3mM CaCl₂; 10 mM HEPES (pH: 7.4)) supplemented with an agonist. Calciummobilization was quantified as the change of peak fluorescence (ΔF) overthe baseline level (F). Data were expressed as the mean S.E. of the ΔF/Fvalue (=RFU, relative fluorescence units) of replicated independentsamples. The analysis was done with the software of the FlexStation®.

Example 6 The Sequence of the First 10 Amino Acids of the MatureDebrilase

The aim of the example is to determine the sequence of the first 10amino acids by N-terminal Edman sequencing. The sample (protein fromFIG. 6 B, lane 5) was successfully sequenced from the N-terminus. Thefollowing major sequence was detected:

IVNGVDTTIQ (SEQ. ID. NO:8)

which corresponds to the mature protein proposed by analysis of theprimary sequence (FIG. 5A).

Example 7 Molecular and Enzymatic Properties of Debrilase

TABLE 1 Summary of molecular and enzymatic properties of Debrilasemolecular weight: 27.4 kDa (pre-pro-peptide), 25.7 kDa (pro-peptide),24.6 kDa (mature protein) K_(m): 0.07 mM* K_(cat): 37.5 · s⁻¹ specificactivity: 90 μmol* · min⁻¹ · mg⁻¹ enzyme pH_(opt): 8 T_(opt): 37° C.*measured in fluorometric assay using Z-Gly-Gly-Arg-AMC as substrate

The invention claimed is:
 1. An isolated nucleic acid molecule encoding(i) a serine protease having the ability to cleave fibrin and casein,said serine protease encoded by (a) a nucleic acid molecule encoding theamino acid sequence of SEQ ID NO: 4; (b) a nucleic acid moleculecomprising or consisting of the nucleotide sequence of SEQ ID NO: 3; (c)a nucleic acid molecule encoding the amino acid sequence which is atleast 90% identical to the amino acid sequence of (a); (d) a nucleicacid molecule comprising or consisting of a nucleotide sequence at least90% identical to the nucleotide sequence of (b); (e) a nucleic acidmolecule which is degenerate with respect to the nucleic acid moleculeof (d); or (f) a nucleic acid molecule corresponding to the nucleic acidmolecule of any one of (a) to (d) wherein T is replaced by U, whereinthe coding sequence of the isolated nucleic acid molecule is operablylinked to a heterologous promoter.
 2. A vector encoding the nucleic acidmolecule of claim
 1. 3. An isolated host cell transformed, transduced ortransfected with the vector of claim
 2. 4. A method of producing aserine protease comprising culturing the host cell of claim 3 andisolating the serine protease, the propeptide or the pre-propeptideproduced.
 5. A serine protease, propeptide, or pre-propeptide encoded bythe nucleic acid molecule of claim 1 or produced by the method of claim4, wherein the serine protease, propeptide, or pre-propeptide isprovided in a pharmaceutical composition with a pharmaceuticallyacceptable sterile carrier.
 6. A fusion protein comprising the serineprotease, the propeptide, or the pre-propeptide of claim 5, wherein thefusion protein is a pre-propeptide having a heterologous signalsequence.
 7. A composition comprising the nucleic acid of claim 1, thevector of claim 2, or the host cell of claim 3 or combinations thereof.8. The composition of claim 7 which is a cosmetic composition.
 9. Thecomposition of claim 7 which is a pharmaceutical composition.
 10. Amethod for treatment of skin peeling, skin smoothening or theintervention with scar formation, comprising contacting skin with thenucleic acid of claim 1, the vector of claim 2, or the host cell ofclaim
 3. 11. A method for treatment of wounds, comprising contacting awound with the nucleic acid of claim 1, the vector of claim 2, or thehost cell of claim
 3. 12. The method of claim 11 wherein the wounds arechronic or slow healing wounds.
 13. A method for treatment of skindiseases accompanied by impaired wound healing comprising contacting askin disease with the nucleic acid of claim 1, the vector of claim 2, orthe host cell of claim
 3. 14. The method claim 10, additionallycomprising administering at least one component selected from the groupof a further protease, nuclease, excipient, anti-microbial agent andpain-relieving agent.
 15. The nucleic acid molecule of claim 1, whereinthe nucleic acid encodes a propeptide of the serine protease of (i), andthe propeptide is encoded by a nucleic acid molecule selected from: (a)a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:6; (b) a nucleic acid molecule comprising or consisting of thenucleotide sequence of SEQ ID NO: 5; (c) a nucleic acid moleculeencoding the amino acid sequence of which is at least 90% identical tothe amino acid sequence of (a); (d) a nucleic acid molecule comprisingor consisting of a nucleotide sequence which is at least 90% identicalto the nucleotide sequence of (b); (e) a nucleic acid molecule which isdegenerate with respect to the nucleic acid molecule of (d); or (f) anucleic acid molecule corresponding to the nucleic acid molecule of anyone of (a) to (d) wherein T is replaced by U.
 16. The nucleic acidmolecule of claim 1, wherein the nucleic acid encodes a pre-propeptideof the serine protease of (i), wherein the pre-propeptide is encoded bya nucleic acid molecule selected from (a) a nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleic acidmolecule comprising or consisting of the nucleotide sequence of SEQ IDNO: 1; (c) a nucleic acid molecule encoding the amino acid sequencewhich is at least 90% identical to the amino acid sequence of (a); (d) anucleic acid molecule comprising or consisting of a nucleotide sequenceat least 90% identical to the nucleotide sequence of (b); (e) a nucleicacid molecule which is degenerate with respect to the nucleic acidmolecule of (d); or (f) a nucleic acid molecule corresponding to thenucleic acid molecule of any one of (a) to (d) wherein T is replaced byU.
 17. The method of claim 11, additionally comprising administering atleast one component selected from the group of a further protease,nuclease, excipient, anti-microbial agent and pain-relieving agent. 18.The method of claim 12, additionally comprising administering at leastone component selected from the group of a further protease, nuclease,excipient, anti-microbial agent and pain-relieving agent.
 19. Theisolated nucleic acid molecule of claim 1, wherein the heterologouspromoter is a yeast promoter.
 20. The isolated nucleic acid molecule ofclaim 19, wherein the heterologous promoter is a methanol induciblepromoter.
 21. The isolated nucleic acid molecule of claim 19, whereinthe heterologous promoter is a yeast promoter selected from an AOX1promoter and a GAL1 promoter.
 22. The isolated nucleic acid molecule ofclaim 1, wherein the serine protease is in the form of a propeptide orpre-propeptide.
 23. The isolated host cell of claim 3, wherein theisolated host cell is a yeast cell.
 24. The isolated host cell of claim23, wherein the yeast cell is of a species selected from Saccharomycescerevisiae, Hansenula polymorpha and Pichia sp.
 25. The isolated hostcell of claim 24, wherein the yeast cell is a Pichia pastoris cell. 26.The serine protease, propeptide, or pre-propeptide of claim 5 which is apropeptide.
 27. The serine protease, propeptide, or pre-propeptide ofclaim 5 which is a propeptide of SEQ ID NO.
 6. 28. The propeptide ofclaim 27, wherein the pharmaceutical composition is suitable for topicaladministration.
 29. The propeptide of claim 27, wherein thepharmaceutical composition is a gel.
 30. The propeptide of claim 29wherein the pharmaceutical composition comprises at least onegel-forming agent selected from cellulose derivatives, vinyl polymers,and carboxypoly-methylene derivatives.
 31. The propeptide of claim 30wherein the pharmaceutical composition comprises at least onegel-forming agent selected from as methyl cellulose, hydroxyethylcellulose, carboxymethyl cellulose, polyvinyl alcohols, polyvinylpyrrolidone, and carbopoline.
 32. The propeptide of claim 29 wherein thepharmaceutical composition comprises at least one gel-forming agentselected from pectins, gums, alginates, agar and gelatin.
 33. Thepropeptide of claim 29 wherein the pharmaceutical composition comprisesone or more auxiliary agents selected from preservatives, antioxidants,stabilizers, colorants and perfumes.
 34. The fusion protein of claim 6,wherein the heterologous sequence is a yeast signal sequence.
 35. Thefusion protein of claim 34, wherein the pre-propeptide comprises thedebrilase propeptide sequence of SEQ ID No. 6 and wherein the nativesignal peptide of debrilase (“MFRFVALFAFVSCALA”) (SEQ. ID. NO 7) issubstituted by the vector encoded-factor signal sequence fromSaccharomyces cerevisiae.